JPH02138287A - Phosphoric ester of novel antibiotic oa-6129 - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R represents H, OH or hydroxysulfonyloxy) exhibiting the following various characteristics. Color reaction; positive to Ehrlich's reagent, chloroplatinic acid reaction and molybdic acid reaction and negative to ninhidrin reaction. Paper chromatography; Rf value 0.03(developing solvent; acetonitrile, HCl and EDTA). High-voltage filter paper electrophoresis: 11-12cm to the anode. USE:An antibacterial agent having strong antibacterial potency and beta-lactamase inhibitory activity. PREPARATION:A material prepared by washing cultured bacteria of Brevibacterium.ammoniagenes (ATCC 6871) is reacted with a substrate of an antibiotic OA-6129 A, B or C expressed by formula II under normal enzymic reaction conditions in the presence of adenosine-5'-triphosphate.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic, and more specifically, to a novel antibiotic containing 7-oxo-1-azabicyclo[3.2.O
] Antibiotics with a hept-2-ene-2-carboxylic acid skeleton generally have high antibacterial activity and β-lactamase inhibitory activity, and have traditionally been synthesized by fermentation, semi-synthesis, and total synthesis methods. 7-oxy-1-azabicyclo[3.2.
0] Hebut-2-ene-2-carboxylic acid derivatives have been produced [for example, Chenamycin (Journal on
Antibiotics, Volume 32 (1979), 1-1
p. 2), evichenamycins (17th Interscience Conference on Antimicrobial Agents and Chemotherapy, Abstracts No. 80 and 81 (1977)), N-acetylchenamycin (West German patent No. 2652681 (1977)
), olivanic acids (Journal of Antibiotics, Vol. 32 (1979), pp. 287-304>
, PS-5 (Journal of Antibiotics, Vol. 32 (1979), pp. 262-286>, PS-
6 (Unexamined Japanese Patent Publication No. 54-59295), PS-7 (Unexamined Japanese Patent Publication No. 54-59295)
4-92983) etc.].

Furthermore, the inventors of the present invention, in part, Streptomyces 0A-6
129 strain (FER) BP-
7-oxo-1-azabicyclo[3.
2.0] It has a bunchyinyl group at the 3-position of the hept-2-ene-2-carboxylic acid skeleton, and an ethyl group, 1-hydroxylethyl group, or 1-hydroxysulfonyloxyethyl group at the 6-position. They discovered that they could produce novel antibiotics that had structural characteristics not described in conventional literature, and named these antibiotics 0A-6129A, B, and C, and filed a patent application earlier. (Unexamined Japanese Patent Publication No. 57-62280,
(See JP-A No. 57-70890 and JP-A No. 57-95987).

The compound of formula (I>) provided by the present invention is obtained by converting the -class hydroxyl group of the antibiotics 0A-6129A, B, and C (hereinafter collectively referred to as "antibiotics 0A-6129J") into a phosphate ester. It is a novel antibiotic that has structural characteristics in that
A-phosphoric acid ester], ■ -hydroxyethyl group-containing compound as "antibiotic 0A-6129B-phosphoric acid ester", and 1-hydroxysulfonyloxyethyl group 5O20H (CH, -CH-) compound as [antibiotic Substance 0A-
6129C-phosphoric acid ester".

In addition, these phosphate esters are referred to as “antibiotic OA-6”.
129 phosphoric acid esters".

The phosphate ester of the novel antibiotic 0A-6129 of formula ('I) is unique in that it is more stable than conventionally proposed antibiotics with the same basic skeleton, and is also described in the above-mentioned known literature. In addition to having strong antibacterial activity and β-lactamase inhibitory activity, it also has the ability to synergistically enhance the antibacterial activity of antibacterial substances such as penicillins and cephalosporins against β-lactamase producing bacteria. It is useful as an antibacterial agent.

The compound of formula (I) can also be in the form of a salt of the carboxyl group and/or the phosphate group at the 2-position; examples of such salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts. Salts: alkaline earth metal salts such as calcium salts and magnesium salts; other metal salts such as aluminum salts; ammonium salts; primary and secondary salts such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine and jetanolamine. Salts with organic bases such as benzathine salts and propylene salts, among which pharmaceutically acceptable salts are preferred, especially sodium salts, karyuno salts, etc.
Alkali metal salts such as salts are preferred.

According to the present invention, the antibiotic 0A-612 of the formula (1)
The phosphoric acid ester of 9 is an antibiotic represented by the formula (n)
Cultured cells of microorganisms that can phosphorylate pantothenic acid in the presence of ATP (adenosine-5'-triphosphate) under normal enzyme reaction conditions using A-6129A, B or C as a substrate, or It can be produced by reacting with a treated product.

The cultured microbial cells or processed products of the microbial cells used in the present invention phosphorylate the primary hydroxyl group of the above-mentioned compound of formula (n) to produce a compound of formula (1) with a corresponding substituent at the 6-position. Bacterial cells or processed microbial cells belonging to any genus can be used as long as they have the ability to phosphorylate pantothenic acid. What you get (e.g. Agricultural
and Biological Chemistry
o! , 36.84-92 (1972))
Among them, washed cultured cells of Brevibacterium ammoniagenes ATCC 6871 are advantageously used.

In the enzymatic reaction using the bacterial cells, ATP is added as a phosphate donor to the buffer solution of p116-8.5 to prepare a solution in which the bacterial cells are suspended, and each of the cells shown by formula (n) is This can be carried out by a method of adding and reacting compounds.

The 12% solution contains phosphoric acid in consideration of the stability of the compound of formula (I[>) and is advantageously adjusted to a range of p116-8,5, particularly 0117-8.

The reaction in such a buffer is generally carried out at a temperature in the range of 20 to 45°C, preferably 30 to 40°C, and the reaction time varies depending on the reaction temperature, the bacterial cells used, or the bacterial cell treatment, but is usually It is in the range of 30 minutes to 10 hours.

Furthermore, in order to suitably carry out the reaction, a surfactant such as higher aliphatic sodium sulfate or a metal ion such as magnesium which can contribute to stabilization of enzyme activity may be present in the reaction solution, if necessary.

The reaction conditions to be used can be determined by a person skilled in the art through simple experiments depending on the characteristics of the bacterial cells used or the treated bacterial cells.
Optimal conditions can be easily determined.

In order to isolate the thus produced compound of formula (I>) from the reaction mixture, after the reaction, the bacterial cells or the bacterial cell product are removed by a separation method known per se, such as filtration, centrifugation, extraction, etc. It is recovered from liquid, supernatant liquid, extract liquid, etc.

Recovery can be carried out by various methods known per se,
In particular, methods often used for the recovery of carboxylic acid type antibiotics are advantageously applied. For example, solvent extraction with ethyl acetate, n-butanol, etc. at low pH and transfer of the solvent layer to a high pH water tank; activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20 ( Adsorption with Mitsubishi Kasei Corporation) etc. and elution with methanol water, acetone water etc.; Dowex 1x
2 (manufactured by Dow Chemical Company), QAE-Sephadex A-
Adsorption and elution with ion exchange resins such as 25 (manufactured by Pharmacia), DEAE-Cellulose Whatman DE-32 (manufactured by Whatman), DEAE-Sephadex A-25 (manufactured by Pharmacia); Sephadex G-10 (manufactured by Pharmacia);
Gel filtration such as Bio Gel P-2 (manufactured by Bio Rat); Cellulose, Avicel SF
Column chromatography (manufactured by American Viscose Company); forced precipitation by adding a solvent such as acetone; freeze-drying; and the like may be used alone or in appropriate combinations, and in some cases repeatedly.

The behavior of the compound of formula (I) during the recovery and purification process can be quantitatively measured by the bioassay method and bioautography described below.

Thus, antibiotic 0A-612 with the properties described above
9A-phosphoric ester, OA-6129B-phosphoric ester or OA-6129C-phosphoric ester are obtained.

The phosphate ester of this antibiotic 0A-6129 is generally more stable in its salt form than in its free form.
It is preferable to treat it in the form of a salt when it is used in the pharmaceutical applications described below, when it is used as an intermediate for further conversion into derivatives, or when it is subjected to the purification process described above.

The conversion of the phosphate ester of antibiotic 0A-6129 into its salt form is carried out according to methods known per se.
This can be done by treating the phosphoric acid ester of No. 6129 with an inorganic or organic base. Inorganic or organic bases that can be used in this salt-forming reaction include, for example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide; alkaline earth metals such as calcium hydroxide and magnesium hydroxide. hydroxides; examples include primary, secondary or tertiary organic amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, jetanolamine, benzathine, and protylene.

The phosphoric acid ester of antibiotic 0A-6129 or its salt provided by the present invention has a wide range of antibacterial activity, and can be used against various microorganisms, such as Staphylococcus, Sarcina, etc.
It exhibits very strong antibacterial activity against positive bacteria belonging to the genus Bacillus, etc., and also exhibits very strong antibacterial activity against, for example, alkaline earth metals and Durham-negative bacteria belonging to the genus Comamonas.

Furthermore, the phosphate ester of the antibiotic 0A-6129 of the present invention exhibits strong antibacterial activity against Durham-negative bacteria belonging to the genus Escherichia, Klebsiella, Proteus, and the like.

In particular, the phosphate ester of the antibiotic 0A-6129 of the present invention is resistant to antibiotics having a β-lactam ring, such as Citrobacter spp., Teus spp., Enterobacter spp., Klebsiella spp., Serratia spp. It has a fairly strong antibacterial activity against Durham-negative bacteria belonging to
It is unique in that it is stable in mammalian kidney homogenates.

The antibacterial spectrum of the phosphate ester of the antibiotic 0A-6129 of the present invention is demonstrated by the measurement of the minimum inhibitory concentration against various pathogenic test bacteria described below (Table 1).

Furthermore, as mentioned above, the phosphate ester of the antibiotic 0A-6129 of the present invention is more stable than conventionally known antibiotics of the same kind, and its stability in mammalian kidney homogenate in particular is as described below. This is evident from the measurement of residual activity in response to various test kidney homogenate treatments (
Table 2).

(Hereinafter in the margin) Table 2 Stability of phosphate ester of antibiotic 0A-6129 and related compounds to various +RF visceral homogenates 4 (Hereinafter in the margin) Next, the present invention will be further explained with reference to Examples. In addition, qualitative and quantitative analyzes of antibacterial active substances used in the following examples were performed by the following methods.

(1) Bioassay method Comamonas terrigen cultured overnight on Nutriendo agar.
a) Suspend B-996 cells in nutrient broth to prepare a seed mother liquor whose 610 nm absorbance derived from the seedlings is 0.04. Far East Powder Bouillon (
Kyokuto Pharmaceutical Industry (manufactured by 11) 0.8% and Bact Agar (
(Manufactured by Difco) 1% seed mother solution on a 1% agar medium
%, and dispensed 7 ml each into 9-diameter Vetri dishes and solidified to prepare a Comamonas assay plate.

(2) Bioautography In the above bioassay method, instead of using a 9-diameter Petri dish, use a dish measuring 32 cm vertically x 24 cm horizontally, and dispense 100 ml of agar medium inoculated with the test bacteria and solidify to create a large assay plate. .

The paper chromatography filter paper after the development of the test solution was pasted on the agar surface of the large assay plate prepared above, removed after 15 minutes, the large assay plate was incubated at 35°C for 20 hours, and the paper chromatography filter was applied from the position of the inhibition zone. It is possible to calculate the Rf value of the filter paper (qualitatively) and semi-quantitatively from the size of the inhibition zone. When using a thin layer chromatography plate, place it on the agar surface with a piece of thin paper so that the component side touches the plate, remove it after 15 minutes, and perform qualitative and semi-quantitative analysis using the same procedure as above.

Example 1 Brevibacterium ammoniagenes (ATCC68
71) Cultivation method Glucose 1%, Hefton 1.5%, K 2 HP O
40.3%, NaC1012%, MgSO4-7H20
0.02%, yeast upper qs 0.1% (pH before sterilization 7.0>
5 g of the medium consisting of the following ingredients was dispensed into 500 ml Erlenmeyer flasks in 100 ml portions and sterilized at 120'C for 15 minutes. After cooling, Brevibacterium ammoniagenes (A
TCC6871) was aseptically inoculated and cultured with shaking at 28°C for 2 days. After culturing, bacterial cells were obtained by centrifugation, -0,
After washing twice with 01M phosphate buffer (9117,1), it was suspended in 100 ml of the same buffer and stored frozen.

Example 2 Preparation of antibiotic 0A-6129A-phosphate ester AT
P (2Na salt) 300mg, Mg504-7H2
Dissolve 0148■ in 5 ml of distilled water and add Na HCO3
After adjusting the pH to around 7, add 1M phosphate buffer (pH 7, 4
) 5 ml, 100 ml of the bacterial cell suspension obtained in Example 1
1. 100 ml of sodium lauryl sulfate was added and stirred well. Add to this the antibiotic OA-6129A (Na)!
>40μ was added and gently shaken at 35°C for 4 hours.

After the reaction, the bacterial cells were removed by centrifugation, and the supernatant was subjected to high-pressure filter paper electrophoresis.Whatman No., IF paper, 1500V/30
cm, 50 minutes, pH 8.6 veronal buffer), there was a slight raw material spot at 4 on on the anode side, 111
A new antibiotic 0A-6129A-phosphate ester spot was observed in the area.

This reaction solution (centrifuged supernatant) was transferred to a QAE-Sephadex A-25 (manufactured by Pharmacia) column (3, OX40) equilibrated in advance with 0.01 M phosphate buffer (O117,4).
cm>, washed with the same buffer, and eluted with a concentration gradient of NaCl dissolved in the same buffer. Antibiotic 0A-
Since 6129A-phosphoric acid ester is eluted at a concentration around 0.251VI, its active fraction was collected and preliminarily adjusted to 0°0
Diaion I-r P-20AG (manufactured by Mitsubishi Chemical Corporation) column equilibrated with 1M phosphoric acid M solution (pH 7, 4)
3, OX40cm). This was eluted with distilled water, and the active fraction was lyophilized to obtain partially purified sample 16■. Dissolve this specimen in 1 ml of distilled water and use Cellulofine GC.
-15-m (manufactured by Chisso) column (1,9X 65cm
0A6129A-phosphate ester was obtained by lyophilization of the active fraction.

This antibiotic 0A-6129A-phosphate ester exhibited the following properties.

(1) Ultraviolet absorption spectrum λ+ measured in 0, 01 M phosphate buffer (pH 7, 4)
aax nm: 302, IF,',: 87.9 (2) Nuclear magnetic resonance spectrum (solvent: D20, internal standard DSS
) δ (ppm): 0.98 (31+, t, J=7.OH2, CH2-CH
3) 1.50-2.00 (2H, m, CH2-cHi
) 2.47 (211t, J=6.511z, NH-CH
2-CH2-Co) 2.70-3.83 (IIH, m,
C-4H2, C-6H, 5-CH.

CH2-NH, NH-CH2-CH2-Co, (3) Color reaction Ehrlich's reagent: Positive chloroplatinic acid reaction: Positive Molybdic acid reaction: Positive Ninhydrin reaction: Negative (4) Paper chromatography Toyo Roshi No. 50 Developing solvent Acetonitrile: 0. IM Tri
5-HC1 buffer (1) H7,5): 0. IM E
DTA=120:30:1 Detection method Comana
Bioautography Rf value using S. terrigena B99G: 0.03 (at this time, antibiotics 0A-6129A,
and the Rf value of antibiotic PS-5 is 0.26.0, respectively.
．． It was 31. ) (5) High pressure filter paper electrophoresis Whatman No. 1P paper veronal buffer (pH 8,6) 1500V 730cm, 50 minutes reverse current extraction method Comamonas terrigena
[Bioautography mobility according to 3996 11-12 cm on the anode side (at this time, antibiotic 0A-6129A is 4-5 cm, PS-5 is 6-7 (1
) Do not move to the anode side.

(6) Behavior towards phosphatase Decomposed by treatment with alkaline phosphatase (Sigma P3877) to produce antibiotic 0A-6129A.

Example 3 Preparation and isolation and purification of antibiotic 0A-6129B-phosphate ester Antibiotic 0A-61291340 was prepared in the same manner as in Example 2.
■ was phosphorylated. After removing the bacterial cells from the reaction solution, 0 and OI were added in advance.
QAE-Sephadex A-25 (manufactured by Pharmacia) column (3
, OX 40 cm). After washing the column with the same buffer, it was eluted with a concentration gradient of NaCl dissolved in the same buffer. Antibiotic 0A-6129B-phosphate ester was eluted around 0.35M, so its active fraction was collected.
Diaion HP-20AG (manufactured by Mitsubishi Chemical Corporation) column (3, OX4) equilibrated with 0.01 M phosphate buffer in advance
0 cm>.

After thorough washing with the same buffer, elution was performed with 10% acetone. The active fraction was collected and concentrated under reduced pressure using a dry ice trap to a volume of about 1 ml, and this was added to Cellulofine GC-1.
Gel filtration was performed using a 5-m (manufactured by Chisso) column (1.9 x 65 cm). Freeze-dry the active fraction to make antibiotic 0A
-6129B-phosphoric acid ester 11.3μ was obtained.

The obtained antibiotic 0A-6129B-phosphate ester exhibited the following properties.

(1) Ultraviolet absorption spectrum λ11 measured in 0.01M phosphate buffer (pH 7, 4)
, nm: 302, E,',: 82.4 (2) Nuclear magnetic resonance spectrum (solvent = D20, internal standard DSS) δ (ppm): 2.47 (2N, t, J = 6.5 Hz, NH- CH2-
CH2-CO) 2.80-4.50 (14 tl, m,
C-4H2, C-5H, C-6H1NH-CH2-CH
, -CO2 (3) Color reaction Ehrlich's reagent: Positive chloroplatinic acid reaction: Positive Molybdic acid reaction: Positive Ninhydrin reaction: Negative (4) Paper chromatography Toyo Roshi No. 50 Developing solvent Acetonitrile: 0. IM Tri 5
-HCli street solution (pH 7,5): 0.1M EDT
A=120:30:1 Detection method comamonas
terrigena B99B Bio-O1 Hepatography Rf value 0.01 (at this time, antibiotic 0A-6129A,
and the Rf values of antibiotic PS-5 are (0, 26,
It was 0.31. ) (5) High pressure filter paper electrophoresis Whatman No. 1 Filter paper veronal buffer (pH 8,6>1500V/30cm, 50 minutes reverse current extraction method Co
Mamonas terrigena B996 Bio-O 1 to Graphie mobility 11 to 12 cm to the anode side (At this time, antibiotic 0A-6129A moved to the anode side by 4 to 5 cm, and PS-5 moved to the anode side by 6 to 71 cm.) (6) Against phosphatase Behavior alkaline phosphatase (Sigma P 3877)
It is degraded upon processing to produce antibiotic 0A-6129B.

Example 4 Preparation of antibiotic 0A-6129C-phosphate ester Antibiotic 0A-6129C1 (potency equivalent) was dissolved in 0.2 ml of M15 phosphate buffer (p117.4) to 100 mM.
ATP solution (adjusted to pH 6-7 with NaHCO3)
Add 0.05 ml of 0.05 ml of 1.100 m M M g S O4 solution and 0.2 ml of the bacterial cell suspension obtained in the same manner as in Example 1, and after stirring, add 0.5 μ of sodium lauryl sulfate. Shake at ℃ for 4 hours. After the reaction, the bacterial cells were removed by centrifugation, and the supernatant was subjected to high-pressure R paper electrophoresis under the following conditions. As a result, there was a small spot of raw material at 6.5 on the anode side and a new spot at 9.41. Na0A-
A spot of 6129C-phosphate ester was observed.

High pressure P paper electrophoresis Whatman No. 1$ paper Veronal buffer (pH 8,6) 1500V/30am, 30 minutes Reverse current extraction method Comamonas terrigena
Bioautography mobility by B996 (all on the anode side) Antibiotic 0A-6129C6-7 n0A-612
9C-phosphate ester 9-10cnlII
OA 6129A 2~3au Noah PS 5
4-5cn Also, antibiotics 0
A-6129C-phosphate ester section was cut out and 0.
After being removed from the tube in 01M phosphate buffer (pH 8,/l) and treated with alkaline phosphatase (Sigma P3877), high-pressure filter paper electrophoresis was performed again, and a spot of antibiotic 0A-6129C was observed.

The method for preparing the antibiotic 0A-6129 substance used as the raw material of the present invention is shown in the following reference example.

Reference Example (A) Put 100 tnl of seed culture medium (S-1) with the following composition into a 500 ml Erlenmeyer flask,
It was sterilized at 120°C for 15 minutes by a conventional method.

On the other hand, Strepitomyces sp. 0A-6129 (S
treptomyces sp, 0A-6129)
After the spores of the bacterial strain were sufficiently attached, the platinum loop was inoculated onto the seed medium and incubated at 28°C for 48 hours on a rotary shaker (
200 rpm, amplitude 7 CR) shaking culture. 200 ml of this seed mother culture solution was added to a seed mother medium with the following composition (SE-4>
151 into a 30.0 volume jar fermentor, stirred at 28°C, 400 rl) and 7.1/m
Aerated agitation culture was performed for 90 hours under aeration conditions. As an antifoaming agent, 0.07% of silicone KM-75 (registered trademark, manufactured by Shin-Etsu Chemical Co., Ltd.) was used.

(B) 2 g of the seed mother culture solution obtained in (A) above after 24 hours of culture was mixed into a production medium with the following composition (GM-1>100.
f! Inoculated into a 200g fermentation tank containing
Aerated agitation culture was performed for 90 hours under conditions of stirring at 200 rpm and aeration of 501/11n. As an antifoaming agent, 0.07% of silicone KM-75 (mentioned above) was used.

The culture solution was sampled over time, and the antibacterial activity of the centrifuged supernatant was measured.

The measurement results at each time were as shown in the table below (
The antibacterial titer is PS-5 sodium salt equivalent titer).

Culture time (hours) Antibacterial titer (μa/m1) 48
2.6 Composition of seed medium (S-1>: Soybean powder yeast extract potato starch Ca CO3 pH (before sterilization) Composition of seed medium (SE-4>) Beef extract Tryptone Glucose soluble starch Yeast extract aCO3 Soybean flour pH (before sterilization) 11.5 24.0 1.5% (W/V) 0.5 No1 2, On O,2n 7.0 0.3% (W/V) 0.5 77 0 .5 II 2.4 n O, 577 Q, 4 11 Q, 5 n 7.5 Composition of production medium (GM-1): Glycerin 8.0% (W/V) Fishmeal
1.0 Tsuno Soybean Flour 3. On CaC0,0,311 to 2 HPO40,2ll Mg5o4' 0.2 ttAdjust NaOH''C'pl-1 to 7.2 before sterilization.

Separately pus, 5 of 0. Dissolved in OIH phosphate buffer,
In addition, 0°0005% (W/V) of vitamin B, 2 was added which was sterilized in an autoclave at 1k (1/cJG) for 5 minutes.

(C) Fermentation solution i after 90 hours of culture obtained in CB) above
Oh,.

5% (W/V) amount of dobucobarite No. 34
(registered trademark, manufactured by Tokai Perlite ■) and separate the bacterial cells using a basket-type centrifuge to obtain a 90ρ culture solution. Adsorb onto a packed column (15 x 100 cm), washed with 5 g of distilled water, and elute with 30% (V/V) acetone water. One fraction was set to 1.0.I!, and the eluate was fractionated. A total of 8.09 eluates from bioassay active fraction 8 to fraction 15 were collected and added to Diaion PA.
306 S (registered trademark, manufactured by Mitsubishi Kasei) Packed column (
8x60cm>, and after washing with 1.0M distilled water,
It was eluted with 3.0% saline. The eluate was fractionated into 500 ml portions, and the eluate containing a total of 5.09 antibiotics 0A-6129A and B from bioassay active fraction 7 to both fractions 16 was collected.

Subsequently, the above-mentioned PA306S packed column was eluted with 30% saline, and the eluate was fractionated into 500 ml portions to obtain a total of 7. The eluate containing Off antibiotic 0A-6129C was collected.

Add 300 g of common salt to the eluate 5.01 containing antibiotics 0A-6129A and B, adsorb it on a Diaion HP-20 packed column (6X 150G+1), and add 5.0 g of distilled water
Total acetone water with concentration increasing linearly from 0% to 40% after washing with 00ml 4. It eluted with O, 1l.

The eluate was fractionated, with each section containing 17 ml. Bioassay active fraction 20 to fraction 130 approximately 1.8
．． The eluate of No. 0 mainly contained antibiotic 0A-6129B and some antibiotic 0A-6129A. Approximately 7 bioassay active fractions from fraction 131 to fraction 170
The eluate of 00ml contained 0A-6129A. The above two sections were each freeze-dried to obtain a brown powder.

Purification of antibiotic 0A-6129A CD) The eluate mainly containing antibiotic 0A-6129A,
The brown powder obtained by freeze-drying is dissolved in a small amount of distilled water, introduced into a Biogel P-2 (registered trademark, manufactured by Biorat) packed column (8 x 100 cm), developed with distilled water, and used for bioassay. The active fraction i, on
t-collected. This active fraction was adsorbed onto a QAE-Sephadex A-25 (registered trademark, manufactured by Pharmacia) packed column (4X40G11) equilibrated with 0.01) monophosphate Sakuragai solution (pH 8,4), The above buffer solution 200
After washing with m+, elution was performed with saline increasing linearly in concentration from 0% to 4% (total 3.0.l). The eluate was fractionated into 15ml portions, bioassay was performed, and fraction 5
A total of 300 ml of active fractions from fraction 1 to fraction 70 were obtained.

Both portions were freeze-dried to obtain a yellowish brown powder.

Dissolve this powder in a small amount of distilled water, add 5g of salt,
Diaion HP-20AG (registered trademark, Mitsubishi Kasei (…
The sample was adsorbed onto a packed column (2×50 cm) manufactured by A. Co., Ltd., and washed with 50 ml of 5% saline, and then with 100 ml of distilled water. The eluate was eluted with acetone water whose concentration increased linearly from 0% to 30% (total 1.01), and the eluate was fractionated using 10 ml of 1 volume. A total of 110 ml of active fractions 35 to 45 was collected by the bioassay and lyophilized to obtain 52 ml of yellow-brown antibiotic 0A-6129A crude powder.

The powder was dissolved in a small amount of distilled water, and Sephadex G-
10 (registered trademark, manufactured by Pharmacia) packed column (2X
80 cm), developed with distilled water, and collected 30 rnl of active volume by bioassay. This active fraction was adsorbed onto a QAE-Sephadex A-25 (described above) packed column (2 x 30 cm) equilibrated with 0.0 IH phosphate buffer (pHB, 7 m), and washed with 50 ml of the above buffer. , elution was carried out with saline solution (total 800 ml) whose concentration increased linearly from 0% to 5%, and the eluate was added to 5 ml.
It was fractionated. A total of 25 ml of active fractions 36 to 40 were collected by bioassay.

Add 4g of salt to both portions and use Diaion HP-20.
AG [previously mentioned] is adsorbed on a packed column (2 x 40 cm),
After washing with 50 ml of distilled water, the eluate was eluted with acetone water (total 800 ml) whose concentration increased linearly from 0% to 30%, each volume was 5 ml, and the eluate was fractionated.

By bioassay, active fractions 105 to 117
A total of 65 ml of water was collected.

By freeze-drying this, antibiotic 0A-6129
21 cm of pale yellow powder of A was obtained.

The obtained freeze-dried specimen of antibiotic 0A-6129A exhibited the following characteristics.

(1) Shape: pale yellow powder (2) Specific rotation: [α] P: 11.6° (Cm1
．． 0, o, oiH phosphate buffer, pH 8, 4) However, in ultraviolet absorption, λ,... The value is when ε of X300 nm is assumed to be 5600.

(3) Molecular formula 1jlj theoretical molecular formula C2oH,,N,,07SNa()?
, W, -479) (4) Ultraviolet absorption spectrum 0,0 IH phosphate buffer (pH 8,4) λ11, nm
(ε) 300 (5600) (5) Infrared absorption spectra] ~ Main peak l of Lamb (K[3r).

pH 11, Cm.

17 (30 (β~lactam) 1660 (amide) 1600 (carboxylate) (6) Nuclear magnetic resonance spectrano\ (solvent: D20) (internal standard: DSS) δ (E) l) m): 1.00 ( 311,t, J=7.511z, CI-
(2'-CI-h) 1.60-2.00 (211, m
, CH2-CH3)2.48(211,t,J
=7.511Z, N-CH2-CH2-CO)2.80
~3.65 (11N, m, C-4H2, C-6HlS
-CH2-CH2-NH,NH-CH2-CH2(E)
Purification of antibiotic 0A-6129B antibiotic 0A-612
The brown powder obtained by freeze-drying the eluate mainly containing 98 was dissolved in a small amount of distilled water, introduced into a column (8 x 100) packed with Biogel P-2 (mentioned above), and washed with distilled water. Develop and bioassay active fraction 1,
I collected ON. This active fraction was preliminarily transferred to a QAE-Sephadex A-25 (mentioned above) packed column (4 x 40 c
After washing with 200 ml of the above buffer, add saline with a concentration increasing linearly from 0% to 4% (total of 3
．． 0. Elution was performed using 1)). The eluate was fractionated into 15 ml portions and subjected to bioassay.
A total of 300 ml of active fraction was obtained.

This powder was dissolved in a small amount of distilled water, 5 g of saline was added, and a Diaion HP-20AG (mentioned above) packed column (
2×50 cm>, washed with 50 ml of 5% saline, and then washed with 100 ml of distilled water. Concentration from 0% to 30
% of acetin water (total 1.O, l
! The eluate was fractionated using 10 ml of eluate. A total of 210 ml of active fractions 15 to 35 were obtained by bioassay. By lyophilizing this fraction, a yellowish brown powder of antibiotic 0A-6129B was obtained. The obtained freeze-dried specimen of antibiotic 0A-6129B had the following characteristics.

(1) Shape: Pale yellow powder (2) Specific rotation: [α] Total 4.7° (c = 1.0, 0.
01M phosphate buffer, pH 8.4) However, in ultraviolet absorption, ε of λ11.3001m is 5
This is the value when it is set to 400.

(3) Molecular formula Theoretical molecular formula C2, H3, N, 0BSNa ()1. W, -
495) (4) Ultraviolet absorption spectrum 0, 018 phosphoric acid Sakuragai solution (pH 8, /l) λ11, nm
(e) 300 (5400) (5) Main peak l of optical external absorption spectrum (K B r ).

□, 011° 1760 (β-lactam) 1660 (amide) 1600 (carboxylate) (6) Nuclear magnetic resonance spec I Heram (solvent: DzO) (internal standard: DSS) δ(1)l)m): 1″i3-shi2.45(2H,t,J=7.0Ilz, N H-C
H2-CH2-CO) 3.94 (IH,s, HO-CH
-CO) (F) Purification of antibiotic 0A-6129C Eluate containing antibiotic 0A-6129C in the first stage 7. O, ll was packed in a Diaion HP-20 (previously mentioned) packed column (5X80
cm) and o,oiH phosphate buffer (1)
118.4)1. After washing with horse mackerel, the eluate was fractionated into 250 ml portions with acetone water (total 6.0 g) whose concentration increased linearly from 0% to 20%, and fractions 9 to 13 were divided into 1.25 N in total. An eluate was obtained.

This eluate was extracted with 1.0 g of methylene chloride containing 3% alkyldimethylbenzyl ammonium chloride [manufactured by Tokyo Kasei (1 Shu)], and then the extracted methylene chloride layer was extracted with 300 g of an 8% aqueous sodium iodide solution.
Re-extraction was performed with m1.

Biogel P-2 (300 ml of the obtained extract was equilibrated in advance with 0.01) monophosphate Sakuragai solution (pl + 8.4)
The mixture was introduced into a packed column (8 x 100 mm), developed with the above buffer solution, bioassay was performed, and 1.2 g of active fraction was collected.

This eluate was added to a Diaion HP-20 packed column (4X
60 cm>, and after washing with 600 ml of distilled water, acetone water whose concentration changes directly from 0% to 10% (
Total 3. O, Q) were eluted. The eluate was fractionated into 15ml portions and subjected to bioassay.
Total up to 1.1.1! An active fraction was obtained. This eluate was adsorbed onto a column (4 x 40 trn) packed with QAE-Sephadex A-25 (mentioned above) equilibrated with 0.0 IH phosphate buffer (pH 8,4), and 500 m
After washing with 1.1, elution was performed with saline increasing linearly in concentration from 0% to 5% (total of 3.0,1.1 ml). The eluate was fractionated into 15ml portions, bioassay was performed, and fraction 1
A total of 420 ml of active fractions from fraction 12 to fraction 139 were obtained. Next, sodium chloride was added to this active fraction to a final concentration of 5%, and the mixture was adsorbed onto a Diaiont (P-20AG (described above)) packed column (3 x 60 cm) and eluted with deionized water. The eluate was fractionated into 15ml portions,
Perform bioassay and obtain active fractions from fraction 31 to fraction 48, totaling 270 ml. This active fraction was freeze-dried to obtain 135 ml of yellowish brown powder.

This powder (135cm) was dissolved in a small amount of distilled water, introduced into a column packed with Sephadex G-10 (mentioned above) (2 x 70 (1)), developed with distilled water, and bioassayed. Good value.

A QAE-Sephadex A-25 packed column (
1 x 30 cm>, and add 800 ml of the above buffer solution.
After washing with water, elute with saline whose concentration increases directly from 0 to 5% (total 2./1.1!).
A total of 286 ml of fractions 118 to 139 was obtained by fractionating each fraction and performing a bioassay.

Add table salt to this so that the final concentration is 5%, and use a Diaion HP-20AG packed column (3 x 60 cm).
The sample was adsorbed onto the membrane and eluted with deionized water. 10 m of eluate
Each fraction was fractionated, and a total of 90 ml of both fractions having maximum ultraviolet absorption at 300 nm was collected.

This was lyophilized and 18■ pale yellow antibiotic 0A-
6129C powder was obtained.

The obtained freeze-dried specimen of antibiotic 0A-6129C showed the following characteristics.

(1) Shape: Pale yellow powder (2) Specific rotation: [α] P 17.4″ (C-0,5
5, O, OIN phosphate buffer, pH 8,2) (3) Elemental analysis value (C20H29N3011S2 Na
2' As 2H20) Calculated value C37, 91%, 8 5.25%, N 6.
63%, 310.12% Analysis value C37.61%, H5.00%, N 6.3
8%, 3 9.52% (4) Molecule i 597.5731 (Molecular formula C2oH
2,N301,52Na(5) Ultraviolet absorption spectrum 0,01) f Phosphate buffer (pH 8,4) λ,,, n
m (ε): 300.5 (7600) (6) Main peak l of optical external absorption spectrum (K B r ).

νl, (ffll + 1750 (β-lactam) 1660-1595 (amide, carboxylate>1
250-1220 (sulfuric acid ester) (7) Nuclear magnetic resonance spectrum (solvent: D 20 ) (internal standard: D
SS) δ (ppm): NH-CH2-CH2-Co, s-c2-c2-NH) 3.83 (111, dd, J=5.5tlz, J=9.
5Hz, C3,94(ltl,s,,HO-CH-C
O) 4.40-4.43 (IH, m, C-5H) 6H
)

Claims (1)

[Claims] (1) Structural formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ In the formula, R represents a hydrogen atom, a hydroxyl group, or a hydroxysulfonyloxy group. Compounds and salts thereof.